The present invention relates to methods and compositions that can be employed to introduce toxins and nucleic acids into the cytoplasm or nucleus of a eukaryotic cell, particularly a cell of a higher vertebrate. The invention allows for the efficient and specific delivery of the toxins and nucleic acids into cells that bind an antibody. The invention particularly concerns the use of a fusion protein of streptavidin and protein A sequences to form a non-covalent complex of a toxin or nucleic acid and an antibody.
The invention provides a method of treatment of human disease by the introduction of toxins or antisense nucleotides into human cells, e.g., tumor cells, in vivo or ex vivo. The invention also provides methods of conducting biological research and methods useful in the production of biological products by introducing exogenous duplex DNA molecules into cultured cells.
The selective introduction of compounds into the cytoplasm or nucleus of specific cells has been a valuable technique in biological and medical research and in medical practice. Cell specific targeting of cytotoxins has been accomplished by complexing toxins with cell binding proteins that can preferentially bind targeted cells. The cell-binding proteins of the complex can be either antibodies, particularly monoclonal antibodies, or protein ligands, e.g., /growth factors/ which recognize the corresponding surface antigens or receptor. Complexes of toxin and antibody have been termed immunotoxins.
Conventionally, the toxin and cell binding protein of the complex have been linked covalently through either chemical coupling or gene fusion. Conventional immunotoxins have been made by chemically linking a toxin component to an antibody, typically a monoclonal antibody, with a heterobifunctional cross-linking reagent that is non-specific. Accordingly, this method yields a heterogeneous product in which some toxin molecules block the antibody""s ability to bind antigen by linking to the F(ab) portion of the antibody. Additionally, the coupling chemistry can partially destroy the toxins activity.
Many of the problems associated with chemical conjugation have been overcome through the generation of single-chain fusion toxins using recombinant DNA technology. However, this technology requires that a new recombinant toxin for each target cell. The biological activity of each new recombinant toxin is unpredictable.
Alternative techniques have been developed in which the cytotoxin is non-covalently linked to the antibody or ligand. One such technique exploits the specific interaction between Staphylococcal aureus protein A and immunoglobulins to generate antibody complexes with two specificities. According to this technique, protein A is complexed with antibodies of two different specificities: a toxin specific antibody and a cell surface specific antibody. Such complexes have been used to deliver ricin toxin into targeted cells (Laky, et al., 1986/1987, Immunology Letters 14:127-132). In a second immunotoxin targeting system, single chain antibodies are fused with streptavidin which has a strong and specific binding affinity for biotin. Using this construct, biotinylated toxin was delivered into a target cell (Dubel, et al., 1995, Journal of Immunological Methods, 178:201-209).
Recently, Sano et al. described a fusion protein consisting of streptavidin and one or two immunoglobulin G (IgG)-binding domains of protein A in Escherichia coli. (U.S. Pat. No. 5,328,985, issued Jul. 12, 1994, which is hereby incorporated by reference in its entirety). The streptavidin-protein A (ST-PA) fusion protein has functional biotin and IgG binding sites. Sano further described complexes of the streptavidin-protein A fusion protein, a monoclonal antibody to BSA, and biotinylated horseradish peroxidase.
Sano also described a method of labeling cells using the ST-PA fusion protein. Cells were incubated with an antibody to a cell surface antigen, Thy-1. The chimeric protein-biotinylated marker complex was subsequently added to the cell suspension. This technique was used to deliver biotinylated FITC to the surface of cells having Thy-1 antigen on their surface. However, Sano did not describe or suggest the use of the ST-PA fusion protein to deliver compounds into the cytoplasm or nucleus of specific cells.
Immunotoxins appear to enter the cell via receptor-mediated endocytosis (Pastan et al., 1986, Cell 47:1-44 and Pirker et al., 1987, Lymphokines 14:361-382). Binding of the antibody moiety of the immunotoxin complex to the surface receptor is followed by, first, clustering of the complex into coated pits and then by internalization of the complex into endosomes or receptosomes within the cell (Middlebrook et al., 1994, Microbiol. Rev., 48:199-221; Morris et al., 1985, Infect. Immun. 50:721-727; Fitzgerald et al., 1980, Cell 21:867-873). During the journey into the cell, the complex may be transported through different intracellular compartments that vary in pH and proteolytic enzyme activity before the toxins are translocated across an intracellular membrane and into the cell cytoplasm where they can cause cell death.
A second area which has been developed concerns methods for introducing nucleic acids into cells. The most widely used methods employ calcium phosphate or DEAE-dextran to promote uptake of nucleic acids. These methods appear to involve the steps of DNA attachment to the cell surface, entry into the cytoplasm by endocytosis, and subsequent transfer into the nucleus. Maniatis, Laboratory Cloning Manual, volume 2, 16.30. Depending upon the cell type, up to 20% of a population of cultured cells can take up DNA using calcium phosphate or DEAE-dextran.
Electroporation is an alternative transfection method in which an electric field is applied to open pores in the cell plasma membrane. DNA appears to enter the cell through these pores.
Liposomes have also been used to introduce nucleic acids into cells. According to this technique, artificial lipid-bilayer vesicles containing cationic and neutral lipids mediate the transfer of DNA or RNA into cells. The mechanism of liposome-mediated transfection, is not well understood, but it appears that negatively charged phosphate groups on DNA bind to the positively charged surface of the liposome, and that the residual positive charge binds to negatively charged sialic acid residues on the cell surface.
Sano did not use the complex to introduce nucleic acid into the cell. Sano et al. described DNA-antibody complexes with the ST-PA fusion protein by incorporating a single biotin molecule at one end of a linearized pUC 19 plasmid. In contrast to the methods of transfecting nucleic acids into the cell which are described above.
The invention relates to a method of delivering toxins or nucleic acids into specific cell types and to the complexes for the practice of the method. According to the invention, an antibody that recognizes a cell surface antigen is non-covalently bound to the antibody binding site of a ST-PA fusion protein; a biotinylated toxin or nucleic acid is bound to the biotin-binding site. In an alternative embodiment, the toxin or nucleic acid can be bound to a third biotinylated molecule, an adapter, which is bound to the biotin binding-site.
In one embodiment of the present invention, a nucleic acid is delivered into a specific cell type. The nucleic acid can be a biotinylated single stranded nucleic acid bound to the biotin binding site of the ST-PA fusion protein. In an alternative embodiment, the nucleic acid can be a duplex nucleic acid that forms a complementary triplex with a biotinylated single stranded nucleic acid, which is in turn bound to the biotin binding site.
The method of the invention relates to the steps of forming a complex between a streptavidin-protein A fusion protein; an antibody, that is specific for a cell surface protein, which undergoes endocytosis after binding with the antibody; and some targeted material e.g. a biotinylated multidrug resistance (mdr) gene product, prodrug, toxin or nucleic acid; isolating the complex from toxin that is not bound to the biotin binding site; and exposing the target cell, to the complex so that the targeted material enters the cell.
3.1 Definitions
As used herein,
toxin, refers to holotoxins, modified toxins, catalytic subunits of toxins, or any enzymes not normally present in a cell that under defined conditions cause the cell""s death. A biotinylated toxin refers to a toxin that is either directly biotinylated or one that is bound to a biotinylated adapter.
The term xe2x80x9cantibodyxe2x80x9d refers to any molecule which contains one or more functional antigen binding domains and an Fc domain that specifically binds protein A.